Sequential C-terminal degradation of peptides and proteins

ABSTRACT

Reagents and method for the C-terminal sequencing of proteins and peptides are disclosed. The reagents include sodium trimethylsilanolate and trimethyl N-oxide. Derivatized, activated polyethylene supports for peptide samples subjected to C-terminal sequencing are described.

This application is a continuation-in-part of Bailey application Ser.No. 07/576,943 filed Aug. 13, 1990, now U.S. Pat. No. 5,059,540, whichin turn is a continuation-in-part of PCT/US90/02723 filed May 18, 1990,each of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the sequential degradation of peptides andproteins from the carboxy-terminus. More particularly, the inventionrelates to a method for the cleavage of the C-terminal thiohydantoinamino acid from the derivatized peptide which results from the use of asilylisothiocyanate as a coupling reagent in carboxy-terminal sequencingprocedures. The invention also relates to derivatized and activatedsupports for covalent immobilization of peptides to be sequenced.

BACKGROUND OF THE INVENTION

A. Background

The development of methods for the sequential degradation of proteinsand peptides from the carboxy-terminus has been the objective of severalstudies. See Ward, C. W., Practical Protein Chemistry - A Handbook(Darbre, A., ed.) (1986) and Rangarajan, M., Protein/Peptide SequenceAnalysis: Current (1988). Such a method would complement existingN-terminal degradations based on the Edman chemistry. Edman, P., Acta.Chem. Scand. 4:283-293 (1950). The most widely studied method andprobably the most attractive because of its similarity to the Edmandegradation has been the conversion of amino acids into thiohydantoins.This reaction, originally observed by Johnson and Nicolet, J. Am. Chem.Soc. 33:1973-1978 (1911), was first applied to the sequentialdegradation of proteins from the carboxy-terminus by Schlack and Kumpf,Z. Physiol. Chem. 154:125-170 (1926). These authors reacted ammoniumthiocyanate, dissolved in acetic acid and acetic anhydride, withN-benzoylated peptides to form carboxyl-terminal1-acyl-2-thiohydantoins. Exposure to strong base was used to liberatethe amino acid thiohydantoin and generate a new carboxyl-terminal aminoacid. The main disadvantages of this procedure have been the severity ofthe conditions required for complete derivatization of the C-terminalamino acid and for the subsequent cleavage of the peptidylthiohydantoinderivative into a new shortened peptide and an amino acid thiohydantoinderivative.

Since this work was published, numerous groups have tried to reduce theseverity of the conditions required, particularly in the cleavage of thepeptidylthiohydantoin, in order to apply this chemistry to thesequential degradation of proteins from the carboxyl terminal end.Lesser concentrations of sodium hydroxide than originally used bySchlack and Kumpf and of barium hydroxide were found to effectivelycleave peptidylthiohydantoins. See Waley, S. G., et al., J. Chem. Soc.1951:2394-2397 (1951); Kjaer, A., et al., Acta Chem. Scand. 6:448-450(1952); Turner, R. A., et al., Biochim. Biophys. Acta. 13:553-559(1954). Other groups used acidic conditions based on the originalprocedure used by Johnson and Nicolet for the de-acetylation of aminoacid thiohydantoins. See Tibbs, J., Nature 168:910 (1951); Baptist, V.H., et al., J. Am. Chem. Soc. 75:1727-1729 (1953). These authors addedconcentrated hydrochloric acid to the coupling solution to causecleavage of the peptidylthiohydantoin bond. Unlike hydroxide which wasshown to cause breakdown of the thiohydantoin amino acids, hydrochloricacid was shown not to destroy the amino acid thiohydantoins. SeeScoffone, E., et al., Ric. Sci. 26:865-871 (1956); Fox, S. W., et al.,J. Am. Chem. Soc. 77:3119-3122 (1955); Stark, G. R., Biochem.7:1796-1807 (1968). Cromwell, L. D., et al., Biochem. 8:4735-4740 (1969)showed that the concentrated hydrochloric acid could be used to cleavethe thiohydantoin amino acid at room temperature. The major drawbackwith this procedure was that when applied to proteins, no more than twoor three cycles could be performed.

Yamashita, S., Biochem. Biophys. Acta. 229:301-309 (1971) found thatcleavage of peptidylthiohydantoins could be done in a repetitive mannerwith a protonated cation exchange resin. Application of this procedureto 100 mmol quantities of papain and ribonuclease was reported to give14 and 10 cycles, respectively, although no details were given. SeeYamashita, S., et al., Proc. Hoshi. Pharm. 13:136-138 (1971). Starkreported that certain organic bases, such as morpholine or piperidine,could be substituted for sodium hydroxide, and along the same lines,Kubo, H., et al., Chem. Pharm. Bull. 19:210-211 (1971) reported thataqueous triethylamine (0.5M) could be used to effectively cleavepeptidylthiohydantoins. Stark appeared to have solved the cleavageproblem by introducing acetohydroxamic acid in aqueous pyridine at pH8.2 as a cleavage reagent. This reagent was shown to rapidly andspecifically cleave peptidylthiohydantoins at room temperature and atmild pH.

Conditions for the formulation of the peptidylthiohydantoins wereimproved by Stark and Dwulet, F. E., et al., Int. J. Peptide and ProteinRes. 13:122-129 (1979), who reported on the use of thiocyanic acidrather than thiocyanate salts, and more recently by the introduction oftrimethylsilylisothiocyanate (TMS-ITC) as a coupling reagent. See Hawke,D. H., et al., Anal. Biochem. 166:298-307 (1987). The use of thisreagent for C-terminal sequencing has been patented. See Hawke U.S. Pat.No. 4,837,165. This reagent significantly improved the yields ofpeptidylthiohydantoin formation and reduced the number of complicatingside products. Cleavage of peptidylthiohydantoins by 12N HCl (Hawke,1987) and by acetohydroxamate (Miller, C. G., et al., Techniques inProtein Chemistry (Hugli, T. E., ed.) pp. 67-68, Academic Press (1989))failed to yield more than a few cycles of degradation. B. The CleavageProblem

Although the cleavage reaction has been extensively studied since thethiocyanate chemistry for C-terminal degradation was first proposed bySchlack and Kumpf in 1926, a chemical method has not yet been proposedthat is capable of an extended degradation. Cleavage in 1N sodiumhydroxide as first proposed by Schlack and Kumpf (1926) is well known tohydrolyze proteins and peptides at other sites in addition to cleavageof the C-terminal peptidylthiohydantoin. The released thiohydantoinamino acid derivatives are also known to be unstable in hydroxidesolutions. Scoffone, supra. Cleavage by hydroxide is known to convertthe side chain amide groups of asparagine and glutamine residues to acarboxylic group making these residues indistinguishable from aspartateand glutamate, respectively.

When cleavage of peptidylthiohydantoins by 12N HCl was applied toproteins and peptides no more than 2 or 3 cycles could be performed.See, Cromwell, supra and Hawke, supra. This was probably due todifferences in the rate of hydrolysis of peptidylthiohydantoinscontaining different amino acid side chains as well as to hydrolysis ofother internal amide bonds. Likewise, during the synthesis of thestandard amino acid thiohydantoin derivatives corresponding to thenaturally occurring amino acids, it was observed that the rate ofdeacetylation of the N-acetylthiohydantoin amino acids by 12 HCldepended on the nature of the amino acid side chain. Bailey, J. M., etal. Biochem. 29:3145-3156 (1990).

Attempts by Dwulet, supra, to reproduce the resin based cleavage methodof Yamashita, supra, was reported to be unsuccessful. Cleavage ofpeptidylthiohydantoins with aqueous methanesulfonic acid was alsoattempted by Dwulet and by Bailey, et al., both without success.Methanesulfonic acid was chosen since it is equivalent to the acidicgroup on the resin employed by Yamashita (1971) and Yamashita, et al.(1971).

Cleavage of the peptidylthiohydantoin derivatives with acetohydroxamateas originally reported by Stark, supra, was found to result in theformation of stable hydroxamate esters at the C-terminus of theshortened peptide (Bailey, et al., supra). Depending on the conditionsemployed, between 684 and 93% of the peptide was derivatized at theC-terminus and thus prevented from further sequencing. Although Stark,supra, predicted such hydroxamate esters to form as an intermediateduring cleavage, it was assumed that they would break down under theconditions used for cleavage or continued sequencing. The peptidylhydroxamate esters formed from cleavage with acetohydroxamate, like thehydroxamate esters studied by Stieglitz, J., et al., J. Am. Chem. Soc.36:272-301 (1914) and Scott, A. W., et al., J. Am. Chem. Soc.49:2545-2549 (1927), are stable under the acidic conditions used forthiohydantoin formation and can only be hydrolyzed to a free peptidylcarboxylic group, capable of continued sequencing, under strongly basicconditions. This probably explains the low repetitive yields of Stark,supra; Meuth, J. L., et al., Biochem. 21:3750-3757 (1982) and Miller,supra, when aqueous acetohydroxamate was employed as a cleavage reagent.

Cleavage of peptidylthiohydantoins by aqueous triethylamine wasoriginally reported by Kubo, H., et al., Chem. Pharm. Bull. 19:210-211(1971), Dwulet, et al., supra, and Meuth, et al., supra. The lattergroup commented on the usefulness of triethylamine as a cleavage reagentfor automated sequencing because of its volatility, but declined topursue this method apparently in favor of cleavage by acetohydroxamate.Cleavage of peptidylthiohydantoins, in the solution phase, by a 2%aqueous solution of triethylamine was found to be rapid (half-times of 1min. and 5 min. at 37° C. and 22° C., respectively) and quantitative,yielding only shortened peptide capable of continued sequencing and theamino acid thiohydantoin derivative. Bailey, et al., supra.

The automation of C-terminal sequencing requires prolonged (1 to 10days) storage of reagents in glass bottles at room temperature withinthe instrument. The reagents used for sequencing must be stable to theseconditions. Storage of triethylamine in water rapidly results in thebreakdown of the triethylazine. These breakdown products include primaryand secondary amines which can subsequently block the shortened peptidefrom further sequencing. Free radical compounds are also formed duringthe breakdown of triethylamine. These free radical compounds are oftenUV absorbing and can interfere with the subsequent HPLC detection of thereleased thiodydantoin amino acid. Applicant's experience with usingaqueous triethylamine (5% triethylamine in water) for automatedC-terminal sequencing has consistently resulted in repetitive yields nohigher than 60%, thereby permitting no more than three cycles ofC-terminal degradation to be performed on peptides covalently coupled toPVDF or polyethylene membrane supports.

C. Peptide Sample Supports

In the preferred practice of the C-terminal sequencing chemistry of thisinvention, the peptide sample is covalently attached to a solid support.Applicant and others (Inglis et al., Met. Protein Sequence Analysis(Jornavall/Hoog/Gustavsson, Eds.) pp. 23-24, Birkhauser-Verlag, Basel(1991); Wittman-Liebold, et al., Met. Protein Sequence Analysis(Jornavall/Hoog/Gustavsson, Eds.) pp. 9-21, Birkhauser-Verlag, Basel(1991); Hawke and Boyd, Met. Protein Sequence Analysis(Jornavall/Hoog/Gustavsson, Eds.) pp. 35-45, Birkhauser-Verlag, Basel(1991)), have recognized that C-terminal chemistry is preferably appliedto samples covalently attached, at the N-terminal, to a solid phase.Covalent immobilization of the sample on a solid support allows the useof reagents and solvents optimal for sequencing without causing samplewashout, the capability to efficiently wash the sample to removereaction by-products which might otherwise interfere with identificationof the released thiohydantoin amino acids, and prevents mechanicallosses associated with most solution phase methods. In general,automated solid phase chemistry is expected to be more efficient andless labor intensive compared to manual solution phase methods.

Since the introduction of the solid-phase approach to N-terminal proteinsequencing by Laursen, R. A. J. Amer. Chem. Soc., 88:5344-5346 (1966),several different types of functionalized supports have been describedfor the covalent immobilization of polypeptide samples. These includepolystyrene resins, polyacrylamide resins, and glass beads substitutedwith aminoalkyl or aminophenyl groups (Laursen and Machleidt, MethodsBiochem. Anal. 26:201-284 (1980); Machleidt, Modern Methods in ProteinChemistry (Tschesche, H., Ed.) pp. 262-302, de Gruyter, Berlin/New York(1983)). Typically these amino functionalized supports are activated forprotein coupling with bifunctional reagents such as phenylenedisothiocyanate (DITC). The DITC group is capable of forming a stablethiourea linkage to the support and the peptide N-terminal amino groupor epsilon amino group of side chain lysines. Recently glass beadsderivatized with isothiocyanato, ainophenyl and aminethylaminopropylgroups (Song-Ping Liang and Laursen, Anal. Biochem. 188:366-373 (1990)),glass fibre sheets functionalized with aminphenyl groups (Aebersold etal., Anal. Biochem. 187:56-65 (1990)), and PVDF (polyvinylidenedifluoride) membranes derivatized with aryl amines and DITC (Pappin etal., Current Research in Protein Chemistry (Villafranca, J. J., Ed.) pp.191-202, Academic Press, Inc. (1990)) have been used for the covalentimmobilization of polypeptides for N-terminal sequencing. Thepolypeptides are either immobilized by coupling between the epsilonamino groups of the lysine and the isothiocyanate groups on the solidsupport using the established DITC chemistry or by the coupling of theactivated C-terminal carboxyl groups of the polypeptides and the aminogroups on the matrix.

Many of the initial studies involving the application of the thiocyanatechemistry for C-terminal sequencing to the solid phase have involved theuse of glass beads for the covalent immobilization of peptide samples(Williams and Kassall, FEBS Lett. 54:353-357 (1975); Rangarajan andDarbre, Biochem. J. 157:307-316 (1976); Meuth et al., Biochem.21:3750-3757 (1982); Hawke et al., Anal. Biochem. 166:298-307 (1987);Inglis, et al. Methods in Protein Sequence Analysis (Wittmann-Liebold,B., Ed.) pp. 137-144, Springer-Verlag (1989). More recent work hasinvolved the use of carboxylic acid modified PVDF (Bailey and Shavely,Techniques in Protein Chemistry: II (Villafranca, J. J., Ed.) pp.115-129, Academic Press, Inc. (1991)), DITC-activated amino PVDF (Milleret al., Techniques in Protein Chemistry (Hugli, T. E., Ed.) pp. 67-78,Academic Press, Inc. (1989), Inglis et al., Met. Protein SequenceAnalysis (Jornval/Hoog/Gustavsson, Eds.) pp. 23-24, Birkhauser-Verlag,Basel (1991)), and a disuccinimidoyl carbonate polyamide resin (Hawkeand Boyd, Met. Protein Sequence Analysis (Jornvall/Hoog/Gustavsson,Eds.) pp. 35-45, Birkhauser-Verlag, Basel (1991).

The use of glass and PVDF supports for C-terminal sequencing is attendedby disadvantages. Siloxane bonds are formed on derivatization of theglass supports. These bonds are base labile resulting in loss of thecovalently coupled peptide sample. The carboxyl modified PVDF and theDITC-activated amino PVDF are both physically and chemically alteredadversely due to dehydro- fluorination (Dias and McCarthy,Macromolecules 17:2529-2531 (1984)) caused by the basic cleavagereagents used during the course of C-terminal sequencing. Thesemembranes may turn successively darker and more brittle on eachC-terminal sequencing cycle.

SUMMARY OF THE INVENTION

This invention provides novel C-terminal peptide cleavage reagents,including (i) alkali metal salts of lower trialkylsilanols and (ii)trialkylamine N-oxides. These novel cleavage reagents have particularutility in, but are not limited to, sequencing procedures in which asilyl isothiocyanate is used as the coupling reagent. Sodium trimethylsilanolate and trimethyl amine N-oxide are preferred. In the preferredpractice of the invention the N-terminal of the peptide sample iscovalently bound to an activated PE-COOH solid phase.

DESCRIPTION OF THE FIGURES

FIG. 1 is the formula of sodium trimethylsilanolate.

FIG. 2 is the formula of trimethylamine N-oxide.

FIG. 3 is the postulated mechanism of the cleavage reaction with sodiumtrimethylsilanolate and the use of aqueous acid, e.g., trifluoraceticacid (TFA), following the cleavage reaction.

FIG. 3A shows an analogous mechanism in which sodium trimethylsilanolate is used following the cleavage reaction.

FIG. 4 shows the sequencing of leucine-enkephalin (YGGFL) covalentlycoupled to PVDF.

FIG. 5 shows the sequencing from the C-terminal of KVILF covalentlyattached to a PVDF membrane.

FIG. 6 shows the sequencing from the C-terminal of the peptide (YGGFL)covalently coupled to an activated PE-COOH membrane.

FIG. 7 shows the sequencing from the C-terminal of the peptide (RGYALG)covalently coupled to an activated PE-COOH membrane.

FIG. 8 shows the sequencing from the C-terminal of the peptide(YGGFMRGL) covalently coupled to an activated PE-COOH membrane.

FIG. 9 shows the sequencing from the C-terminal of the peptide (YGGFL)covalently coupled to a linker 11-amino undecaonic acid.

FIG. 10 is a schematic of the instrument used for C-terminal sequencing.

DETAILED DESCRIPTION OF THE INVENTION

The mechanism of acid and base hydrolysis of acylthiohydantoins wasstudied in detail by Congdon and Edward, Can. J. Chem. 50:3767-3788(1972) and a number of cleavage reagents were tested by Stark, supra.Stark found that oxygen containing nucleophiles were the best choice ofreagents to effect this reaction. Although acetohydroxamate is anexcellent cleavage reagent for the first amino acid, it forms a stablepeptidyl hydroxamate ester, which is difficult to remove, and whicheffectively blocks the shortened peptide from further sequencing. Thisreagent also results in a high UV absorbing background during subsequentHPLC identification of the released thiohydantoin amino acids. It seemsthat in general any carbon based reagent that is a good nucleophile andthus a good cleavage reagent will also be a poor leaving group, therebyblocking much of the shortened peptide from further sequencing.

Ideally, a cleavage reagent should possess the followingcharacteristics: (1) it should be able to cleave peptidylthiohydantoinsin a volatile, water miscible organic solvent, thus eliminating theproblems of incompatibility of PVDF membranes with water; (2) thereaction should be rapid and specific; (3) the shortened peptide shouldbe capable of continued degradation; (4) the released thiohydantoinamino acid should not be destroyed by this reagent; and (5) this reagentshould not absorb light in the same range as is used for detection ofthe released thiohydantoin amino acid derivatives. Sodiumtrimethylsilanolate (FIG. 1), commercially available from Petrarch(Huls), in, e.g., alcoholic solvents, and trimethyl amine N-oxide (FIG.2) commercially available from Aldrich Chemical co. in alcoholic and awider range of solvents seem to possess all of these characteristics.Cleavage of peptidylthiohydantoins in the solution phase with a 0.05Msolution of sodium trimethylsilanolate in 100% methanol ortrimethylsilylethanol is complete in less than 5 minutes. A 0.05Msolution of sodium trimethylsilanolate in methanol ortrimethylsilylethanol effects cleavage of peptidylthiohydantoins both inthe solution phase and in the solid phase in less than 5 minutes.

More specifically, this invention contemplates the use of the cleavagereagents of this invention in a concentration of from about 0.025 molarto about 0.25 molar, preferably from about 0.1 to about 0.2 molar, inmethanol, trimethylsilylethanol or similar alcohols or, in the case oftrimethylamine N-oxide, a wider range of solvents.

The most probable mechanism of this cleavage involves formation of anunstable C-terminal trimethylsilyl ester which, in the presence of wateror alcohol, rapidly reforms the desired C-terminal carboxylic group. SeeFIG. 3 which illustrates the use of aqueous trifluoroacetic acid (TFA)following the cleavage reaction. Preferably, the concentration of TFA inthis step is from about 0.01M to about 0.2M. The use of TFA or anequivalent acid at this stage significantly facilitates cleavage.

In its more broad aspects, the invention includes cleavage reagentshaving the formula R₃ Sio⁻ X⁺, in which R is a straight or branchedchain hydrocarbon radical having from about 1 to about 10 carbon atomsand X is an alkali metal ion, preferably a sodium or a potassium ion.

The broader aspects of the invention also include trimethylamineN-oxides in which the alkyl groups have from one to about four carbonatoms. Triethyl, tripropyl, triisopropyl, tributyl or triisobutyl amineN-oxides may be used. Such reagents have the formula (R₁)₃ N⁺ -O⁻, inwhich R₁ is an alkyl group having from one to about four carbon atoms.

Suitable supports include activated carboxylic acid modified non-porouspreferably polyethylene films or membrane. This support for C-terminalsequencing, hereinafter generally identified as PE-COOH, offers a numberof advantages over the existing supports. These include: (1) stabilityof the support to the conditions employed for C-terminal sequencing; (2)the ability, due to the hydrophilic nature of the surface groups, to useboth aqueous and organic solvents for performing chemistry on covalentlycoupled polypeptide samples; (3) a high capacity (3.2 nmoles/mm² ofsurface area) to covalently couple polypeptides; (4) the convenient size(1×5 mm) of the support needed for sequencing is similar to thatemployed in applicant's continuous flow reactor for automated N-terminalsequencing.

The PE-COOH films useful as supports in the present invention aretypically 1 mil thick with a range of from 0.5 to 20 mils thick. Thefilms are non-porous and naturally hydrophobic. A preferred method forpreparing PE-COOH from polyethylene films is described in detail in U.S.Pat. No. 4,339,473, which is incorporated herein by reference. ThesePE-COOH films are commercially available from Pall Corporation, 30 SeaCliff Avenue, Glen Cove, N.Y. 11542 under the tradename PERMION.

PE-COOH such as the "PERMION" product of Pall Corporation is activatedby the formation of an ester derivative with which the N-terminus of thepeptide sample may be coupled. Appropriate activation reagents include1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI),1-ethyl-3-(3-dimethylamiopropyl) carbodiimide hydrochloride (EDC),benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP), 1,3-Diisopropylcarbodiimide (DICD).

In general, activation of PE-COOH is accomplished by reaction for anappropriate time, e.g. about two hours, with a solution of an activatingagent in a suitable solvent at about room temperature.

Table I is illustrative.

                  TABLE I                                                         ______________________________________                                        Effect of Different Coupling Reagents                                         And Solvents on the Yield of                                                  Leucine Enkephalin Coupled to PE-COOH                                         Activating                                                                            nmol covalently coupled to membrane (% yield)                         Reagent 50% acetonitrile  50% DMF                                             ______________________________________                                        DCC     0.2 (4.4)         1.8 (39.6)                                          CDI     0.07 (1.5)        0.23 (5.1)                                          EDC     0.32 (6.9)        0.12 (2.6)                                          BOP     0.58 (12.7)       0.29 (6.4)                                          DICD    0.68 (15.0)        0.57 (12.6)                                        ______________________________________                                         DCC  1,3dicyclohexylcarbodiimide                                              CDI  1,1' carbonyldiimidazole                                                 EDC  1ethyl-3-(3-dimethylamiopropyl) carbodiimide hydrochloride               BOP  benzotriazol1-yl-oxy-tris(dimethylamino) phosphonium                     hexafluorophosphate                                                           DICD  1,3Diisopropylcarbodiimide                                         

A strip of PE-COOH (1 cm×0.1 cm) was placed in a 1.5 cm continuous flowreactor (CFR) (Shavely, et al., 1987) sealed at one end. The membraneswere reacted with 30 μl (30 mmol) of the activating reagent for 30 min.at room temperature. The solvent used in all cases was 100% DMF, exceptfor when EDC was the activating agent, when water was the solvent. Afterthe activation reaction, excess reagent was washed away with the solventused for the activation reaction (3 ml). Each sample was then washedwith acetonitrile (1 ml) and dried in a vacuum centrifuge. The activatedmembranes were then reacted with a 30 μl solution (4.54 nmol) of thepeptide, YGGFL, in either 50% aqueous DMF or 50% aqueous acetonitrile ina CFR reactor (sealed at one end) for 16 hours. The membranes werewashed with either 50% acetonitrile or 50% DMF, followed byacetonitrile, and then dried in a vacuum centrifuge. The amount ofcoupled peptide was determined by amino acid compositional analysis ofthe derivatized membrane.

Table II reports the effect of activation time and coupling time on theyield of covalent attachment of YGGFL.

                  TABLE II                                                        ______________________________________                                        Effect of Activation Time and Coupling                                        Time on the Yield of Covalent Attachment of YGGFL                             Activation                                                                              Coupling    Amount Coupled                                          Time (hrs)                                                                              Time (hrs)  (nmol)       Yield                                      ______________________________________                                        0.5       1           0.54          4.0                                       0.5       2           2.11         15.6                                       0.5       4           3.33         24.7                                       0.5       20          5.19         38.4                                       1.0       1           1.01          7.5                                       1.0       2           1.59         11.8                                       1.0       4           2.73         20.2                                       1.0       20          5.57         41.3                                       2.0       1           2.44         18.1                                       2.0       2           2.45         18.1                                       2.0       4           5.25         38.9                                       2.0       20          7.2          53.3                                       4.0       1           0.52          3.9                                       4.0       2           0.52          3.9                                       4.0       4           3.4          25.2                                       4.0       20          4.1          30.4                                       ______________________________________                                    

The membranes (1×12.5 mm) were activated by an excess of DCC inanhydrous DMF (1 g/1 ml) for 2 hours. At the end of the activationreaction, the excess reagent was removed with anhydrous DMF and theactivated membrane strip dried in a vacuum centrifuge. Each activatedmembrane was inserted into a continuous flow reactor (CFR) (Shavely etal., Anaylytical Biochemistry (1987) 163, 517-529) containing a 100 μlsolution of leucine enkephalin (13.5 nmol) in 50% aqueous DMF for theindicated time at 22° C. The microbore tubing on one end of the CFR wassealed by first heating and then pinching closed with pliers. After thecoupling reaction, the membrane was rinsed with coupling solvent andacetonitrile, and then dried in a vacuum centrifuge. The amount ofcovalently coupled peptide was determined by amino acid analysis.

An important aspect of the invention, preferred for the C-terminalsequencing of peptides consisting of a large number of amino acidresidues entail the addition of a linker arm, of the general formula NH₂(CH₂)n COCH, wherein n is between 4 and 10 to the PE-COOH carboxylsfollowed by covalent attachment of the peptide sample to the linker armcarboxyl. Where linker arms are utilized the PE-COOH carboxyls arepreferably activated with 2-fluoro-1-methylpyridine to facilitateattachment to the linker molecule. See generally Mukaiyama, AngewandteChemie 18:707-721 (1979). The linker carboxyls are activated in themanner described for PE-COOH carboxyls.

EXPERIMENTAL PROCEDURES

Materials. Acetic anhydride was purchased from Fisher Chemical Co.Trimethylsilylisothiocyanate (TMS-ITC), anhydrous dimethylformamide(DMF),Benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexaflurophosphate(BOP), 1,3-Diisopropylcarbodiimide (DIIC), and 1,1'-Carbonyldiimidazole(CDI) were from Aldrich. Water was purified on a Millipore Milli Qsystem. All of the peptides used in this study were either obtained fromBachem or Sigma. 1,3-Dicylcohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and triethylamine(sequanal grade) were obtained from Pierce. The carboxylic acid modifiedpolyethylene membranes (PE-COOH) were provided by the Pall Corporation(Long Island, N.Y.). The amino acid thiohydantoins used in this studywere synthesized as described (Bailey and Shavely, Biochemistry29:3145-3156 (1990).

Covalent Coupling of Peptides to Carboxylic Acid Modified PVDF. A pieceof PVDF-COOH (1 cm×0.1 cm) was placed in a continuous flow reactor (CFR)(Shavely, et al. Anal. Biochem. 163:517-529 (1987)), 2 μl of1,3-dicyclohexylcarbodiimide (DCC) (2 mmol/ml) in N,N-dimethylformamide(DMF) was added through a Hamilton syringe. The microbore tubing on oneend of the CFR was sealed by first heating and then pinching closed withpliers. The CFR was then placed in a sealed eppendorf tube. After a twohour activation (25° C.), the activated membrane was rinsed with 2 ml ofDMF and then dried under a stream of argon. Leucine enkephalin (2 μl ofa 14 nmol/μl solution in DMF) was carefully layered on the dry membraneand allowed to react overnight. The membrane was washed with methanol (5ml) and dried under a stream of argon. The yield of coupled peptide wasdetermined, after acid hydrolysis of the derivatized membrane, by aminoacid composition analysis. Typical yields were approximately 50%.

Covalent Coupling of Peptides to Carboxylic Acid Modified Polyethylene.A piece of PE-COOH (1×12.5 mm) was placed in a continuous flow reactor(CFR) (Shavely, et al., (1987) supra), and activated an excess of1,3-dicylohexylcarbodiimide in anhydrous DMF (1 g/ml) for 2 hours. Themicrobore tubing on one end of the CFR was sealed by first heating andthen pinching closed with pliers. The CFR was then placed in a sealedeppendorf tube. At the end of the activation reaction, the excessreagent was washed off with anhydrous DMF and dried in a vacuumcentrifuge. Each activated membrane was again inserted into a continuousflow reactor (CFR) (Shavely, et al., (1987) supra) containing thepeptide solution of interest and incubated overnight (20 hours) at 22°C. The capacity of the CFR used was 100 μl. After the coupling reaction,the membrane was rinsed with coupling solvent and acetonitrile, and thendried in a vacuum centrifuge.

Use of this new reagent for cleavage in a 1:1 solution of methanol andtert-butanol as a solvent has allowed the automated sequencing ofseveral peptides covalently coupled to a support such as a polyethyleneor PVDF membrane. Examples are shown for the peptides, KVILF, YGGFMRGL,YGGFL, and RGYALG. In this case the thiohydantoin of arginine co-eluteswith the only major background peak. The combination of methanol withtert-butanol is the preferred solvent for the cleavage reaction. Otheralcoholic solvents useful in the invention include alkanols having fromone to six atoms, e.g. ethanol, isopropanol, 2-trimethylsilylethanol andisobutanol.

EXEMPLIFICATION OF THE C-TERMINAL SEQUENCING INVENTION

The examples described herein illustrate C-terminal sequencingaccomplished with a modified N-terminal sequencer based on the design ofHawke, et al., Anal. Biochem. 147:315-330 (1985), in which a continuousflow reactor as described in Shavely U.S. patent application Ser. No.072,754 was utilized. FIG. 9 is the schematic of the instrument used toperform all of the examples. Release of thiohydantoin amino acids wasdetected by an on-line HPLC. See Shively, Anal. Biochem. 163:517-529(1987). The reagent and solvent delivery system were all constructedfrom perfluoro-elastomers. Delivery valves as generally described inU.S. Pat. No. 4,530,586 provided with electromagnetically actuatedsolenoids and zero dead volume were connected in a modular fashionproviding multiple input to a single output line. The valves whichcontrol the flow of reagents and solvents are computer operated pursuantto a program appropriate for the chemistry involved.

Table III is a program summary for the C-terminal sequencer illustratedby FIG. 9 and used to perform the experiment described in the Example.

                  TABLE III                                                       ______________________________________                                        C-Terminal Sequencer Program Summary                                          Continuous Flow  Conversion     Duration                                      Reactor (CFR)(65° C.)                                                                   Flask (CF)(55° C.)                                                                    (sec)                                         ______________________________________                                        S1 reaction                     120                                           S1 reaction                     120                                                            R4 rinse       120                                           Dry              Dry            100                                           R1 reaction                     180                                           R2 reaction                     450                                           S4 rinse                         60                                           R1 reaction                     180                                           R2 reaction                     450                                           S4 rinse                         60                                           R1 reaction                     180                                           R2 reaction                     450                                           S4 rinse                         60                                           R1 reaction                     180                                           R2 reaction                     450                                           S4 rinse                        120                                           S2 rinse                         60                                           S4 rinse                        120                                           S3 rinse                        120                                           S4 rinse                        120                                           R3 reaction                     1200                                          R3 to CF                         45                                           R3 reaction      Dry            240                                           R3 reaction      Dry            240                                           S2 delivery                      12                                           S2 reaction                     120                                           S2 to CF                         45                                           S2 delivery                      11                                           S2 reaction                     120                                           S2 to CF                         45                                                            inject          7                                            pause            pause           60                                           ______________________________________                                    

EXAMPLE 1 Sequencing of Leucine Enkephalien (YGGFL) (1.2 nmoL)Covalently Coupled to PVDF-COOH

The composition of the reagents and solvents is set forth in Table IV.

                  TABLE IV                                                        ______________________________________                                        Composition of Reagents and Solvents                                          ______________________________________                                        R1       Acetic anhydride                                                     R2       30% TMS-ITC in acetonitrile                                          R3       0.05M Sodium trimethylsilanolate in                                           2-trimethylsilylethanol                                              R4       Methanol                                                             S1       Acetonitrile                                                         S2       0.8% Trifluoroacetic acid in water                                   S3       Methanol                                                             S4       --                                                                   ______________________________________                                    

Activated PVDF-COOH understood to be described in U.S. Pat. No.5,611,861, was obtained from Pall Corporation. The thiohydantoin aminoacid derivatives were separated by reverse phase HPLC. This separationwas performed on Phenomenex Ultracarb 5 ODS(30) column (2.0 mm×25 mm) ona Beckman System Gold with a Shimadzu (SPD-6A) detector. The column waseluted for 2 min with solvent A (0.1% trifluoroacetic acid in water) andthen followed by a discontinuous gradient to solvent B (80%acetonitrile, 10% methanol in water) at a flow rate of 0.15 mL/min at22° C. The gradient used was as follows: 0% B for 2 min, 0-6% B over 3min, 6-35% B over 35 min, 35-50% B over 10 min, and 50-0% B over 10 min.

FIG. 4 depicts the HPLC analysis of products of the reaction.

EXAMPLE 2 Sequencing of KVILF (1.2 nmol) Covalently Coupled to PVDF-COOH

The composition of the reagents and solvents is set forth in Table V.

                  TABLE V                                                         ______________________________________                                        Composition of Reagents and Solvents                                          ______________________________________                                        R1     Acetic anhydride                                                       R2     30% TMS-ITC in acetonitrile                                            R3     0.05M Sodium trimethylsilanolate in methanol                           R4     Methanol                                                               S1     Acetonitrile                                                           S2     0.8% Trifluoroacetic acid in water                                     S3     --                                                                     S4     --                                                                     ______________________________________                                    

The thiohydantoin amino acid derivatives were separated by reverse phaseHPLC. This separation was performed on Phenomenex Ultracarb 5 ODS(30)column (2.0 mm×25 mm) on a Beckman System Gold with a Shimadzu (SPD-6A)detector. The column was eluted for 2 min with solvent A (0.1%trifluoroacetic acid in water) and then followed by a discontinuousgradient to solvent B (80% acetonitrile, 10% methanol in water) at aflow rate of 0.15 mL/min at 22° C. The gradient used was as follows: 0%B for 2 min, 0-6% B over 3 min, 6-354 B over 35 min, 35-50% B over 10min, and 50-0% B over 10 min.

FIG. 5 depicts the HPLC analysis of products of the reaction.

EXAMPLE 3 Sequencing of Leucine Enkephalin (YGGFL) (24.2 nmol)Covalently Coupled to Polyethylene

The composition of the reagents and solvents is set forth in Table VI.

                  TABLE VI                                                        ______________________________________                                        Composition of Reagents and Solvents                                          ______________________________________                                        R1      Acetic anhydride                                                      R2      30% TMS-ITC in acetonitrile (v/v)                                     R3      0.10M Sodium trimethylsilanolate in 50%                                       methanol, 50% t-butyl alcohol                                         R4      Methanol                                                              S1      5% Triethylamine in water                                             S2      1.0% Trifluoroacetic acid in water                                    S3      50% Methanol, 50% Water                                               S4      Acetonitrile                                                          ______________________________________                                    

The thiohydantoin amino acid derivatives were separated by reverse phaseHPLC. The separation was performed on a Phenomenex Ultracarb 5 ODS(30)column (2.0 mm×25 mm) on a Beckman 126 Pump Module with a Shimadzu(SPD-6A) detector. The column was eluted for 5 min with solvent A(0.006% phosphoric acid, 0.006% triethylamine, 0.045% pentanesulfonicacid) and then followed by a discontinuous gradient to solvent B (0.03%phosphoric acid, 0.0454 triethylamine, 30% acetonitrile, 0.045%pentanesulfonic acid) at a flow rate of 0.15 mL/min at 22° C. Thegradient used was as follows: 0% B for 5 min, 0-20% B over 7 min,20-1004 B over 25 min, 100% for 23 min, and 100-0% B over 2 min.

FIG. 6 depicts the results of the HPLC analyses.

EXAMPLE 4 Sequencing of RGYALG (8.1 nmol) Covalently Coupled to PE-COOH

The sequencing of RGYALG was performed exactly as described in Example3.

FIG. 7 depicts the HPLC analyses.

EXAMPLE 5 Sequencing of YGGFMRGL (9.6 nmol) Covalently Coupled toPE-COOH

The sequencing of YGGFMRGL was performed exactly as described in Example4.

FIG. 8 depicts the HPLC analyses.

EXAMPLE 6 Sequencing of YGGFL (3.7 mol) Covalently Coupled to the Linker11-Aminoundecanoic Acid H₂ N(CH₂)₁₀ CO₂ H

FIG. 9 depicts the sequencing from the C-terminal of the peptide YGGFL(3.7 mol) covalently coupled to the linker 11-aminoundecaonic acid H₂ N(CH₂)₁₀ CO₂ H. The linker was attached to PE-COOH by activation of thePE-COOH carboxyl groups with DCC as described. After attachment, thelinker carboxyl groups were similarly activated with DCC and the peptidethen covalently attached. In the absence of linkers after six cycles ofsequencing, 50% of the N-terminal amino acid, Y, is still covalentlyattached when sequencing the peptide YGGFL (FIG. 6). When the samepeptide is sequenced after attachment to the above mentioned linker(FIG. 10), using the same sequencing protocol, only 12% of theN-terminal amino acid, Y is still covalently attached.

The sequencing of YGGFL was performed exactly as described in Example 3.

What is claimed is:
 1. A method for sequencing a peptide by carboxylterminal degradation which comprises coupling the carboxyl terminus of apeptide with a coupling reagent to form a peptidylthiohydantoinderivative and cleaving the peptidylthiohydantoin derivative with areagent having the formula R₃ Sio⁻ X⁺, in which R is a straight orbranched chain hydrocarbon radical having from about 1 to about 10carbon atoms and X is an alkali metal ion to provide a thiohydantoinderivative of the amino acid previously at the carboxyl terminus of apeptide and a peptidyl residue lacking such an amino acid, theN-terminal of said peptide being covalently coupled to an activated,carboxylic acid modified, polyethylene membrane.